Fluorescent dyes and methods of use thereof

ABSTRACT

Provided are methods for labeling target molecules, such as nucleic acids, with fluorescent dye compounds having the formula 
                         
One method embodiment includes contacting reactive group Z of the fluorescent dye compound with the target molecule such that reactive group Z reacts with the target molecule to form a covalent bond between the group and the target molecule. Another method embodiment includes contacting a fluorescent dye compound that further includes a first member of a binding pair, with a target molecule that includes a second member of the binding pair. Also provided are target molecules labeled with the fluorescent dye compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/270,456filed Oct. 11, 2011, which is hereby incorporated by reference in itsentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 8, 2016, isnamed ENZ-100-DI-Application-SL.txt and is 869 bytes in size.

FIELD OF THE INVENTION

The present application generally relates to fluorescent dyes. Morespecifically, the invention is directed to rhodamine and fluoresceindyes useful for labeling nucleic acids and other molecules.

BACKGROUND OF THE INVENTION

Numerous rhodamine and fluorescein dyes are available that are usefulfor labeling nucleic acids, proteins and other molecules. See, e.g.,U.S. Pat. Nos. 6,184,379 and 6,552,199; European Patent Publications 0543 333 and 0 567 622, and references cited therein.

Labeling methods for attaching rhodamine and fluorescein dyes and othernon-radioactive compounds to various molecules are well developed.Non-radioactive labeling methods were initially developed to attachsignal-generating groups onto proteins. This was achieved by modifyinglabels with chemical groups such that they would be capable of reactingwith the amine, thiol, and hydroxyl groups that are naturally present onproteins. Examples of reactive groups that were used for this purposeinclude activated esters such as N-hydroxysuccinimide esters,isothiocyanates and other compounds. Consequently, when it becamedesirable to label nucleotides and nucleic acids by non-radioactivemeans, methods were developed to convert nucleotides and polynucleotidesinto a form that made them functionally similar to proteins. Forinstance, U.S. Pat. No. 4,711,955 discloses the addition of amines tothe 8-position of a purine, the 5-position of a pyrimidine and the7-position of a deazapurine. The same methods that could add a label tothe amine group of a protein could thus be applied towards thesemodified nucleotides.

Dyes have been synthesized with arms containing functional groups withiodoacetamide, isothiocyanate or succinimidyl esters that react withsulfhydryl groups on proteins (Ernst et al., 1989; Mujumdar, et al.,1989; Southwick, et al., 1990). Another series of modified dyes containa sulfonate group on the phenyl portion of an indolenine ring thatincreased the water solubility of the dyes (Mujumdar et al., 1993).Those dyes were activated by treatment with disuccinimidyl carbonate toform succinimidyl esters that were then used to label proteins bysubstitution at the amine groups. Other activating groups have also beenplaced on dyes. U.S. Pat. Nos. 5,627,027 and 5,268,486 describe dyeswhich comprise isothiocyanate, isocyanate, monochlorotriazine,dichlorotriazine, mono or di-halogen substituted pyridine, mono ordi-halogen substituted diazine, aziridine, sulfonyl halide, acid halide,hydroxy-succinimide ester, hydroxy-sulfosuccinimide ester, imido esters,glyoxal groups, aldehydes or other groups, all of which can form acovalent bond with an amine, thiol or hydroxyl group on a targetmolecule.

U.S. Pat. No. 6,110,630 describes cyanine dyes prepared with a series ofreactive groups derived from N-hydroxynaphthalimide. These groupsinclude hydroxysuccinimide, para-nitrophenol, N-hydroxyphtalimide andN-hydroxynaphtalimide, all of which can react with nucleotides modifiedwith primary amines. The same chemical reactions described above werealso described in U.S. Pat. No. 6,114,350 where the constituents wherereversed. There, the cyanine dyes were modified with amine, sulfhydrylor hydroxyl groups and the target molecules were modified to comprisethe appropriate reactive groups.

Labeled nucleotides have been used for the synthesis of DNA and RNAprobes in many enzymatic methods including terminal transferaselabeling, nick translation, random priming, reverse transcription, RNAtranscription and primer extension. Labeled phosphoramidite versions ofthese nucleotides have also been used with automated synthesizers toprepare labeled oligonucleotides. The resulting labeled probes arewidely used in such standard procedures as northern blotting, Southernblotting, in situ hybridization, RNAse protection assays, DNA sequencingreactions, DNA and RNA microarray analysis and chromosome painting.

There is an extensive literature on chemical modification of nucleicacids by means of which a signal moiety is directly or indirectlyattached to a nucleic acid. Primary concerns of this art have been (a)which site in a nucleic acid is used for attachment, i.e. sugar, base orphosphate or analogues thereof, and whether these sites are disruptiveor non-disruptive (see, e.g., U.S. Pat. Nos. 4,711,955 and 5,241,060);(b) the chemistry at the site of attachment that allows linkage to areactive group or signaling moiety that can comprise a spacer groupusually consisting of a single aromatic group (U.S. Pat. Nos. 4,952,685and 5,013,831) or a carbon/carbon aliphatic chain to provide distancebetween the nucleic acid and the reactive group or signaling moiety anda reactive group at the end of the spacer, such as an OH, NH, SH or someother group that can allow coupling to a signaling moiety; and (c) thenature of the signaling moiety.

Although the foregoing have all been descriptions of the various aspectsthat are concerned with the synthesis of modified nucleotides andpolynucleotides, they have also been shown to be significant factorswith regard to the properties of the resultant nucleotides andpolynucleotides. Indeed, there have been numerous demonstrations thatthe modified nucleotides described in the present art have shortcomingscompared to unmodified nucleotides. These factors can have a majorimpact on the ability of these modified nucleotides to be incorporatedby polymerases. A consequence of this is that when using a modified baseas the sole source of that particular nucleotide, there may be a loss inthe amount of nucleic acid synthesis compared to a reaction withunmodified nucleotides. As a result, modified nucleotides are oftenemployed as part of a mixture of modified and unmodified versions of agiven nucleotide. Although this restores synthesis to levels comparableto reactions without any modified nucleotides, a bias is often seenagainst the use of the modified version of the nucleotide. As such, thefinal proportion of modified/unmodified nucleotide may be much lowerthan the ratio of the reagents at the beginning of the reaction. Usersthen have a choice of either using nucleic acids that are minimallylabeled or of decreased yields. When comparable modified nucleotides areused that only comprise a linker arm attached to a base (such asallylamine dUTP) difficulties with incorporation are seldom seen. Assuch, the foregoing problem is likely to be due to the interactions ofthe label with either the polymerase or the active site where synthesisis taking place.

Difficulties in the use of polymerases can be bypassed by the use ofoligonucleotide synthesizers where an ordered chemical joining of e.g.,phosphoramidite derivatives of nucleotides can be used to producelabeled nucleic acids of interest. However, the presence of signalagents on modified nucleotides can still be problematic in this system.For instance, a phosphoramidite of a modified nucleotide may display aloss of coupling efficiency as the chain is extended. Although this maybe problematic in itself, multiple and especially successive use ofmodified nucleotides in a sequence for a synthetic oligonucleotide canresult in a drastic cumulative loss of product. Additionally, chemicalsynthesis is in itself not always an appropriate solution. There may becircumstances where labeled nucleic acids need to be of larger lengthsthan is practical for a synthesizer. Also, an intrinsic part ofsynthetic approaches is a necessity for a discrete sequence for thenucleic acid. For many purposes, a pool or library of nucleic acidswould require an impractically large number of different species forsynthetic approaches.

An example of a method to increase the yield of labeled oligonucleotidesor polynucleotide is to use a non-interfering group such as anallylamine modified analogue during synthesis by either a polymerase oran oligonucleotide synthesizer. Labeling is then carried outpost-synthetically by attachment of the desired group through thechemically reactive allylamine moieties. However, in this case, althoughincorporation or coupling efficiency may be restored, there may still beproblems of the coupling efficiencies of attachment of the desired groupto the allylamine. For instance, coupling of labels to allylaminemoieties in a nucleic acid is dramatically less efficient fordouble-stranded DNA compared to single-stranded targets. In addition topotential yield problems, the functionality of the modification may beaffected by how it is attached to a base. For instance if a hapten isattached to a base, the nature of the arm separating the hapten from thebase may affect its accessibility to a potential binding partner. When asignal generating moiety is attached through a base, the nature of thearm may also affect interactions between the signal generating moietyand the nucleotide and polynucleotide.

Attempts to limit these deleterious interactions have been carried outin several ways. For instance, attachment of the arm to the base hasbeen carried out with either a double bond alkene group (U.S. Pat. No.4,711,955) or a triple bond alkyne group (U.S. Pat. No. 5,047,519)thereby inducing a directionality of the linker away from the nucleotideor polynucleotide. In addition, deleterious interactions can be limitedby having the arm displace the active or signal group away from thenucleotide or polynucleotide by lengthening the spacer group. Forinstance, a commercially available modified nucleotide includes a sevencarbon aliphatic chain (Cat. No. 42724, ENZO Biochem, Inc. New York,N.Y.) between the base and a biotin moiety used for signal generation.This product was further improved by the substitution of linkers with 11or even 16 carbon lengths (Cat. Nos. 42722 and 42723, ENZO Biochem, Inc.New York, N.Y.). A comparison was also carried out using differentlength linker arms and a cyanine dye labeled nucleotide (Zhu et al.,1994). A direct improvement in efficiency was noted as the length wasincreased from 10 to 17 and from 17 to 24.

Another approach was taken in U.S. Pat. No. 5,948,648, which describesthe use of multiple alkyne or aromatic groups connecting a marker to anucleotide.

It is noted that the above-described difficulties do not occur with theuse of polymerases with labeled probes (e.g., labeled phosphoramiditeprobes), where the probes are extended along a template using unmodifiednucleotides or derivatives, since the polymerase does not encounter thelabel-modified nucleotide during the extension reaction. Thus, probesthat are utilized in extension reactions and are synthesized chemicallycan employ a greater variety of conjugation methods and linkers thanoligonucleotides or polynucleotides that are labeled enzymatically.

Amplification of nucleic acids from clinical samples has become a widelyused technique. The first methodology for this process, the PolymeraseChain Reaction (PCR), is described in U.S. Pat. No. 4,683,202. Sincethat time, other methodologies such as Ligation Chain Reaction (LCR)(U.S. Pat. No. 5,494,810), GAP-LCR (U.S. Pat. No. 6,004,286), NucleicAcid Sequence Based Amplification (NASBA) (U.S. Pat. No. 5,130,238),Strand Displacement Amplification (SDA) (U.S. Pat. Nos. 5,270,184 and5,455,166) and Loop Mediated Amplification (U.S. Pat. No. 6,743,605;European Patent Publication 0 971 039) have been described. Detection ofan amplified product derived from the appropriate target has beencarried out in number of ways. In PCR as described in U.S. Pat. No.4,683,202, gel analysis was used to detect the presence of a discretenucleic acid species. Identification of this species as being indicativeof the presence of the intended target was determined by size assessmentand the use of negative controls lacking the target sequence. Theplacement of the primers used for amplification dictated a specific sizefor the product from appropriate target sequence. Spurious amplificationproducts made from non-target sequences were unlikely to have the samesize product as the target derived sequence. Alternatively, moreelaborate methods have been used to examine the particular nature of thesequences that are present in the amplification product. For instance,restriction enzyme digestion has been used to determine the presence,absence or spatial location of specific sequences. The presence of theappropriate sequences has also been established by hybridizationexperiments. In this method, the amplification product can be used aseither the target or as a probe.

The foregoing detection methods have historically been used after theamplification reaction was completed. More recently, methods have beendescribed for measuring the extent of synthesis during the course ofamplification, i.e. “real-time” detection. For instance, in the simplestsystem, an intercalating agent is present during the amplificationreaction (U.S. Pat. Nos. 5,994,056 and 6,174,670). This method takesadvantage of an enhancement of fluorescence exhibited by the binding ofan intercalator to double-stranded nucleic acids. Measurement of theamount of fluorescence can take place post-synthetically in afluorometer after the reaction is over, or real time measurements can becarried out during the course of the reaction by using a PCR cyclermachine that is equipped with a fluorescence detection system and usescapillary tubes for the reactions (U.S. Pat. Nos. 5,455,175 and6,174,670). As the amount of double-stranded material rises during thecourse of amplification, the amount of signal also increases. Thesensitivity of this system depends upon a sufficient amount ofdouble-stranded nucleic acid being produced to generate a signal that isdistinguishable from the fluorescence of a) unbound intercalator and b)intercalator molecules bound to single-stranded primers in the reactionmix. Specificity is derived from the nature of the amplificationreaction itself or by looking at a T_(m) profile of the reactionproducts. Although the initial work was done with ethidium bromide, SYBRGreen™ is more commonly used at the present time. A variation of thissystem is described in U.S. Pat. No. 6,323,337, where the primers usedin PCR reactions were modified with quenchers thereby reducing signalgeneration of a fluorescent intercalator that was bound to a primerdimer molecule. Signal generation from target derived amplicons couldstill take place since amplicons derived from target sequences comprisedintercalators bound to segments that were sufficiently distant from thequenchers.

Another method of analysis that depends upon incorporation is describedin U.S. Pat. No. 5,866,336. In that system, signal generation isdependent upon the incorporation of primers into double-strandedamplification products. The primers are designed such that they haveextra sequences added onto their 5′ ends. In the absence ofamplification, stem-loop structures are formed through intramolecularhybridization that consequently bring an energy transfer (FRET) quencherinto proximity with an energy donor thereby preventing fluorescence.However, when a primer becomes incorporated into double-strandedamplicons, the quencher and donor become physically separated and thedonor is then able to produce a fluorescent signal. The specificity ofthis system depends upon the specificity of the amplification reactionitself. Since the stem-loop sequences are derived from extra sequences,the T_(m) profile of signal generation is the same whether the ampliconswere derived from the appropriate target molecules or from non-targetsequences.

In addition to incorporation based assays, probe based systems can alsobe used for real-time analysis. For instance, a dual probe system can beused in a homogeneous assay to detect the presence of appropriate targetsequences. In this method, one probe comprises an energy donor and theother probe comprises an energy acceptor (European Patent Publication 0070 685). Thus, when the target sequence is present, the two probes canbind to adjacent sequences and allow energy transfer to take place. Inthe absence of target sequences, the probes remain unbound and no energytransfer takes place. Even if by chance there are non-target sequencesin a sample that are sufficiently homologous that binding of one or bothprobes takes place, no signal is generated since energy transferrequires that both probes bind and that they be in a particularproximity to each other. Advantage of this system has been taken in U.S.Pat. No. 6,174,670 for real time detection of PCR amplification usingthe capillary tube equipped PCR machine described previously. The primerannealing step during each individual cycle can also allow thesimultaneous binding of each probe to target sequences providing anassessment of the presence and amount of the target sequences. In afurther refinement of this method, one of the primers comprises anenergy transfer element and a single energy transfer probe is used.Labeled probes have also been used in conjunction with fluorescentintercalators to allow the specificity of the probe methodology to becombined with the enhancement of fluorescence derived from binding tonucleic acids. This was first described in U.S. Pat. No. 4,868,103 andlater described with amplification reactions in PCT Publication WO99/28500.

Probes have also been used that comprise an energy donor and an energyacceptor in the same nucleic acid. In these assays, the energy acceptor“quenches” fluorescent energy emission in the absence of appropriatecomplementary targets. In one system described in U.S. Pat. No.5,118,801, “molecular beacons” are used where the energy donor and thequencher are kept in proximity by secondary structures with internalbase pairing. When the target sequences are present, complementarysequences in the molecular beacons allow hybridization events thatdestroy the secondary structure thereby allowing energy emission. Inanother system that has been termed Taqman, use is made of thedouble-stranded selectivity of the exonuclease activity of Taqpolymerase (U.S. Pat. No. 5,210,015). When target molecules are present,hybridization of the probe to complementary sequences converts thesingle-stranded probe into a substrate for the exonuclease. Degradationof the probe separates an energy transfer donor from the quencherthereby releasing light from the donor.

U.S. Patent Publication 2005/0137388 also describes various formats forutilization of FRET interactions for various nucleic acid assays.

Because fluorescent dyes are used widely, e.g., for labeling nucleicacids, proteins and other molecules, there is an ongoing need for newdyes to provide more options for labeling methods and linker armselections, spectral profiles and energy transfer (FRET) pair selection.The present invention addresses that need.

SUMMARY OF THE INVENTION

In some embodiments, compounds are provided that comprise:

wherein

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are independently H, F, Cl,Br, I, CN, nitro, azido, hydroxyl, amino, hydrazino, (substituted) aryl,(substituted) aroxyl, alkenyl, alkynyl, alkyl, alkoxy, alkylamino,dialkylamino, arylamino, diarylamino, alkyl(aryl)amino, alkanoylamino,alkylthio, alkylcarbonyl, aryl carbonyl, alkylthiocarbonyl,arylthiocarbonyl, alkyloxycarbonyl, aroxycarbonyl, alkylaminocarbonyl,arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl,alkyl(aryl)aminocarbonyl, arylcarboxamido, or Q, the alkyl or alkoxyportions of which are saturated or unsaturated, linear or branched,unsubstituted or further substituted by F, Cl, Br, I, CN, OH, alkenyl,alkynyl, alkylcarbonyl, amide, thioamide, or Q; wherein Q comprises acarboxyl group (CO₂ ⁻), a carbonate ester (COER¹¹), a sulfonate ester(SO₂ER¹¹), a sulfoxide (SOR¹¹), a sulfone (SO₂CR¹¹R¹²R¹³), a sulfonamide(SO₂NR¹¹R¹²), a phosphate (PO₄ ⁼), a phosphate monoester (PO₃ ⁻ER¹¹), aphosphate diester (PO₂ER¹¹ER¹²), a phosphonate (PO₃ ⁼), a phosphonatemonoester (PO₂ ⁻ER¹¹), a phosphonate diester (POER¹¹ER¹²), athiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂ ⁻ER¹¹), athiophosphate diester (PSOER¹¹ER¹²), a thiophosphonate (PSO₂ ⁼), athiophosphonate monoester (PSO⁻ER¹¹), a thiophosphonate diester(PSER¹¹ER¹²), a phosphonamide (PONR¹¹R¹²NR¹⁴R¹⁵), a phosphonamidethioanalogue (PSNR¹¹R¹²NR¹⁴R¹⁵), a phosphoramide (PONR¹¹R¹²NR¹³NR¹⁴R¹⁵),a phosphoramide thioanalogue (PSNR¹¹R¹²NR¹³NR¹⁴R¹⁵), a phosphoramidite(PO₂R¹⁴NR¹¹R¹²) or a phosphoramidite thioanalogue (POSR¹⁴NR¹¹R¹²), whereE can be independently O or S, and where the aryl portions of any of theabove are optionally substituted by F, Cl, Br, I, CN, OH, alkenyl,alkynyl, alkylcarbonyl, amide, or thioamide;

-   -   wherein R¹ in combination with R², R³ in combination with R⁴, R⁵        in combination with R⁶, or R⁹ in combination with R¹⁰ can        independently form a 5-10 member ring structure which is        saturated or unsaturated, and which is optionally further        substituted with an alkyl, an aryl, an alkenyl, an alkynyl, an        alkoxy, an aroxyl, a hydroxyl, an F, a Cl, a Br, an I, a CN, a        nitro, an alkylsulfonyl, an arylsulfonyl, an alkylsulfinyl, an        arylsulfinyl, a (thio)carbonyl, a (thio)carboxylic acid, a        (thio)carboxylic acid ester, a nitro, an amino, a (thio)amide,        an azido, a hydrazino, or a (thio)phosphonate where each alkyl        group or alkoxy group is independently saturated or unsaturated,        linear or branched, or substituted or unsubstituted and each        aryl group wherein is independently optionally substituted with        an F, a Cl, a Br, an I, a CN, an OH, an alkyl, an alkenyl, an        alkynyl, an alkoxy, an aryoxy, an alkylthio, an arylthio, a        nitro, an azido, a hydrazino, a carboxyl, a thiocarboxyl, a        carbonyl, a thiocarbonyl, a carboxylic acid ester, a        thiocarboxylic acid ester, or an unsubstituted or substituted        amino, amide, thioamide, or Q;

R¹¹, R¹², R¹³, R¹⁴ and R¹⁵ are independently a hydrogen, a halogen, anamino group, an alkyl group wherein said alkyl group is saturated orunsaturated, linear or branched, or substituted or unsubstituted, analkoxy group wherein said alkoxy group is saturated or unsaturated,branched or linear, or substituted or unsubstituted, an aryl groupwherein said aryl group is unsubstituted or substituted; wherein R¹¹ incombination with R¹², R¹⁴ in combination with R¹⁵, R¹¹ in combinationwith R¹³, R¹¹ in combination with R¹⁴, R¹² in combination with R¹⁵, orR¹³ in combination with R¹⁴ can independently form a 5-10 member ring;

X is O, OR¹⁶, NR¹⁷R¹⁸ or N⁺R¹⁷R¹⁸; Y is O, OR¹⁶, NR¹⁹R²⁰ or N⁺R¹⁹R²⁰,wherein R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are independently H, alkyl, alkenyl,alkynyl, or aryl; or R¹⁷ in combination with R¹⁸, or R¹⁹ in combinationwith R²⁰ can independently form a 5-10 member ring structure which isoptionally further substituted with alkyl, alkenyl, alkynyl, aryl,alkoxy, F, Cl, Br, I, carboxylic acid or carboxylic acid ester, wherethe alkyl group is saturated or unsaturated, linear or branched, and isoptionally further substituted by F, Cl, Br, I, CN, OH, alkenyl,alkynyl, nitro, azido, hydrazino, alkoxy, aryoxy, alkylthio, arylthio,thiocarboxyl, carbonyl, thiocarbonyl, thiocarboxylic acid ester,unsubstituted or substituted amino, amide, thioamide, or Q, and the arylgroup wherein is optionally substituted by F, Cl, Br, I, CN, OH, alkoxy,aryoxy, alkylthio, arylthio, nitro, azido, hydrazino, carboxyl,thiocarboxyl, carbonyl, thiocarbonyl, carboxylic acid ester,thiocarboxylic acid ester, unsubstituted or substituted amino, amide,thioamide, or Q;

-   -   wherein R¹⁷ in combination with R⁶, R¹⁸ in combination with R⁷,        R¹⁹ in combination with R⁸, or R²⁰ in combination with R⁹, can        independently form a 5- to 10-member ring structure that is        saturated or unsaturated and optionally further substituted with        an alkyl, an aryl, an alkenyl, an alkynyl, an alkoxy, an aroxyl,        a hydroxyl, an F, a Cl, a Br, an L a CN, a nitro, a carbonyl, a        thiocarbonyl, a thiocarboxylic acid, a thiocarboxylic acid        ester, a nitro, an amino, a (thio)amide, an azido, a hydrazino,        or Q, wherein the alkyl group herein is saturated or        unsaturated, linear or branched, substituted or unsubstituted,        an alkoxy group wherein the alkoxy group is saturated or        unsaturated, branched or linear, substituted or unsubstituted;        and wherein the aryl group is optionally substituted with F, Cl,        Br, I, CN, OH, alkenyl, alkynyl, alkoxy, aryoxy, alkylthio,        arylthio, nitro, azido, hydrazino, carboxyl, thiocarboxyl,        carbonyl, thiocarbonyl, carboxylic acid ester, thiocarboxylic        acid ester, unsubstituted or substituted amino, amide,        thioamide, or Q;

A is O, S or NR²¹, wherein R²¹ is a hydrogen, an alkyl, an aryl, analkenyl, an alkynyl, an alkylcarbonyl, an arylcarbonyl, analkylaminocarbonyl, or an arylaminocarbonyl, the alkyl or aryl portionsof which is optionally substituted by an alkyl, an aryl, an alkenyl, analkynyl, an F, a Cl, a Br, an I, a CN, an OH, an alkoxy, an aryoxy, analkylthio, an arylthio, a nitro, an azido, a hydrazino, a thiocarboxyl,a carbonyl, a thiocarbonyl, a thiocarboxylic acid ester, or anunsubstituted or substituted amino, amide, thioamide, or Q.

B is an alkyl, an alkenyl, an alkynyl, or an aryl linker, the alkyl oraryl portions of which is optionally substituted by an alkyl, analkenyl, an alkynyl, an aryl, an F, a Cl, a Br, an I, a CN, an OH, analkoxy, an aryoxy, an alkylthio, an arylthio, a nitro, an azido, ahydrazino, a carboxyl, a thiocarboxyl, a carbonyl, a thiocarbonyl, acarboxylic acid ester, a thiocarboxylic acid ester, or an unsubstitutedor substituted amino, amide, thioamide, or Q; or

-   -   B in combination with A form an amide, a thioamide, a carboxylic        acid ester, a carboxylic acid thioester, a thiocarboxylic acid        ester, an imine, a hyrazone, or Q; and

Z is a reactive group comprising an isocyanate, an isothiocyanate, amonochlorotriazine, a dichlorotriazine, a 4,6-dichloro-1,3,5-triazines,a mono- or di-halogen substituted pyridine, a mono- or di-halogensubstituted diazine, a maleimide, a haloacetamide, an aziridine, asulfonyl halide, a carboxylic acid, an acid halide, a phosphonyl halide,a phosphoramidite (PO₂R¹⁴NR¹¹R¹²), a phosphoramidite thioanalogue(POSR¹⁴NR¹¹R¹²), a hydroxysuccinimide ester, a hydroxysulfosuccinimideester, an imido ester, an azido, a nitrophenol ester, an azide, a3-(2-pyridyl dithio)-propionamide, a glyoxal, an aldehyde, a thiol, anamine, a hydrazine, a hydroxyl, a terminal alkene, a terminal alkyne, aplatinum coordinate group or an alkylating agent.

In other embodiments, a fluorescent dye comprising the above compound isprovided.

Also provided is a fluorescence energy transfer system, comprising theabove-described fluorescent dye and a second dye wherein the second dyeis capable of energy transfer with the fluorescent dye.

Further provided is a kit for labeling a target molecule. The kitcomprises the above-described fluorescent dye with additional reagentsuseful for labeling the target molecule.

A target molecule labeled with the above-described fluorescent dye isalso provided.

Additionally, a method of labeling a target molecule is provided. Themethod comprises contacting reactive group Z of the above-describedfluorescent dye with the target molecule such that reactive group Zreacts with the target molecule to form a covalent bond between reactivegroup Z and the target molecule.

Another method of labeling a target molecule is also provided. Themethod comprises contacting the above-described fluorescent dye, wherethe fluorescent dye further comprises a first member of a binding pair.In this method, the target molecule comprises a second member of thebinding pair.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are graphs showing spectral properties of Dye 1 (EZRed620)and prior art dye LC Red 640 (Roche).

FIG. 1A shows the UV-Vis spectra of LC Red 640 and EZRed620.

FIG. 1B shows the emission spectra of LC Red 640 and EZRed620.

FIG. 1C shows the UV-Vis and emission spectra of LC Red 640.

FIG. 1D shows the UV-Vis and emission spectra of EZRed620.

DETAILED DESCRIPTION

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Additionally, the use of“or” is intended to include “and/or”,unless the context clearly indicates otherwise.

Provided herein are novel rhodamine dyes that are useful for, e.g.,labeling nucleic acids or other molecules. In some embodiments, thepresent invention provides a compound comprising:

wherein

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are independently H, F, Cl,Br, I, CN, nitro, azido, hydroxyl, amino, hydrazino, (substituted) aryl,(substituted) aroxyl, alkenyl, alkynyl, alkyl, alkoxy, alkylamino,dialkylamino, arylamino, diarylamino, alkyl(aryl)amino, alkanoylamino,alkylthio, alkylcarbonyl, aryl carbonyl, alkylthiocarbonyl,arylthiocarbonyl, alkyloxycarbonyl, aroxycarbonyl, alkylaminocarbonyl,arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl,alkyl(aryl)aminocarbonyl, arylcarboxamido, or Q, the alkyl or alkoxyportions of which are saturated or unsaturated, linear or branched,unsubstituted or further substituted by F, Cl, Br, I, CN, OH, alkenyl,alkynyl, alkylcarbonyl, amide, thioamide, or Q; wherein Q comprises acarboxyl group (CO₂ ⁻), a carbonate ester (COER¹¹), a sulfonate ester(SO₂ER¹¹), a sulfoxide (SOR¹¹), a sulfone (SO₂CR¹¹R¹²R¹³), a sulfonamide(SO₂NR¹¹R¹²), a phosphate (PO₄ ⁼), a phosphate monoester (PO₃ ⁻ER¹¹), aphosphate diester (PO₂ER¹¹ER¹²), a phosphonate (PO₃ ⁼), a phosphonatemonoester (PO₂ ⁻ER¹¹), a phosphonate diester (POER¹¹ER¹²), athiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂ ⁻ER¹¹), athiophosphate diester (PSOER¹¹ER¹²), a thiophosphonate (PSO₂ ⁼), athiophosphonate monoester (PSO⁻ER¹¹), a thiophosphonate diester(PSER¹¹ER¹²), a phosphonamide (PONR¹¹R¹²NR¹⁴R¹⁵), a phosphonamidethioanalogue (PSNR¹¹R¹²NR¹⁴R¹⁵), a phosphoramide (PONR¹¹R¹²NR¹³NR¹⁴R¹⁵),a phosphoramide thioanalogue (PSNR¹¹R¹²NR¹³NR¹⁴R¹⁵), a phosphoramidite(PO₂R¹⁴NR¹¹R¹²) or a phosphoramidite thioanalogue (POSR¹⁴NR¹¹R¹²), whereE can be independently O or S, and where the aryl portions of any of theabove are optionally substituted by F, Cl, Br, I, CN, OH, alkenyl,alkynyl, alkylcarbonyl, amide, or thioamide;

-   -   wherein R¹ in combination with R², R³ in combination with R⁴, R⁵        in combination with R⁶, or R⁹ in combination with R¹⁰ can        independently form a 5-10 member ring structure which is        saturated or unsaturated, and which is optionally further        substituted with an alkyl, an aryl, an alkenyl, an alkynyl, an        alkoxy, an aroxyl, a hydroxyl, an F, a Cl, a Br, an L a CN, a        nitro, an alkylsulfonyl, an arylsulfonyl, an alkylsulfinyl, an        arylsulfinyl, a (thio)carbonyl, a (thio)carboxylic acid, a        (thio)carboxylic acid ester, a nitro, an amino, a (thio)amide,        an azido, a hydrazino, or a (thio)phosphonate where each alkyl        group or alkoxy group is independently saturated or unsaturated,        linear or branched, or substituted or unsubstituted and each        aryl group wherein is independently optionally substituted with        an F, a Cl, a Br, an I, a CN, an OH, an alkyl, an alkenyl, an        alkynyl, an alkoxy, an aryoxy, an alkylthio, an arylthio, a        nitro, an azido, a hydrazino, a carboxyl, a thiocarboxyl, a        carbonyl, a thiocarbonyl, a carboxylic acid ester, a        thiocarboxylic acid ester, or an unsubstituted or substituted        amino, amide, thioamide, or Q;

R¹¹, R¹², R¹³, R¹⁴ and R¹⁵ are independently a hydrogen, a halogen, anamino group, an alkyl group wherein said alkyl group is saturated orunsaturated, linear or branched, or substituted or unsubstituted, analkoxy group wherein said alkoxy group is saturated or unsaturated,branched or linear, or substituted or unsubstituted, an aryl groupwherein said aryl group is unsubstituted or substituted; wherein R¹¹ incombination with R¹², R¹⁴ in combination with R¹⁵, R¹¹ in combinationwith R¹³, R¹¹ in combination with R¹⁴, R¹² in combination with R¹⁵, orR¹³ in combination with R¹⁴ can independently form a 5-10 member ring;

X is O, OR¹⁶, NR¹⁷R¹⁸ or N⁺R¹⁷R¹⁸; Y is O, OR¹⁶, NR¹⁹R²⁰ or N⁺R¹⁹R²⁰,wherein R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are independently H, alkyl, alkenyl,alkynyl, or aryl; or R¹⁷ in combination with R¹⁸, or R¹⁹ in combinationwith R²⁰ can independently form a 5-10 member ring structure which isoptionally further substituted with alkyl, alkenyl, alkynyl, aryl,alkoxy, F, Cl, Br, I, carboxylic acid or carboxylic acid ester, wherethe alkyl group is saturated or unsaturated, linear or branched, and isoptionally further substituted by F, Cl, Br, I, CN, OH, alkenyl,alkynyl, nitro, azido, hydrazino, alkoxy, aryoxy, alkylthio, arylthio,thiocarboxyl, carbonyl, thiocarbonyl, thiocarboxylic acid ester,unsubstituted or substituted amino, amide, thioamide, or Q, and the arylgroup wherein is optionally substituted by F, Cl, Br, I, CN, OH, alkoxy,aryoxy, alkylthio, arylthio, nitro, azido, hydrazino, carboxyl,thiocarboxyl, carbonyl, thiocarbonyl, carboxylic acid ester,thiocarboxylic acid ester, unsubstituted or substituted amino, amide,thioamide, or Q;

-   -   wherein R¹⁷ in combination with R⁶, R¹⁸ in combination with R⁷,        R¹⁹ in combination with R⁸, or R²⁰ in combination with R⁹, can        independently form a 5- to 10-member ring structure that is        saturated or unsaturated and optionally further substituted with        an alkyl, an aryl, an alkenyl, an alkynyl, an alkoxy, an aroxyl,        a hydroxyl, an F, a CI, a Br, an I, a CN, a nitro, a carbonyl, a        thiocarbonyl, a thiocarboxylic acid, a thiocarboxylic acid        ester, a nitro, an amino, a (thio)amide, an azido, a hydrazino,        or Q, wherein the alkyl group herein is saturated or        unsaturated, linear or branched, substituted or unsubstituted,        an alkoxy group wherein the alkoxy group is saturated or        unsaturated, branched or linear, substituted or unsubstituted;        and wherein the aryl group is optionally substituted with F, Cl,        Br, I, CN, OH, alkenyl, alkynyl, alkoxy, aryoxy, alkylthio,        arylthio, nitro, azido, hydrazino, carboxyl, thiocarboxyl,        carbonyl, thiocarbonyl, carboxylic acid ester, thiocarboxylic        acid ester, unsubstituted or substituted amino, amide,        thioamide, or Q;

A is O, S or NR²¹, wherein R²¹ is a hydrogen, an alkyl, an aryl, analkenyl, an alkynyl, an alkylcarbonyl, an arylcarbonyl, analkylaminocarbonyl, or an arylaminocarbonyl, the alkyl or aryl portionsof which is optionally substituted by an alkyl, an aryl, an alkenyl, analkynyl, an F, a CI, a Br, an I, a CN, an OH, an alkoxy, an aryoxy, analkylthio, an arylthio, a nitro, an azido, a hydrazino, a thiocarboxyl,a carbonyl, a thiocarbonyl, a thiocarboxylic acid ester, or anunsubstituted or substituted amino, amide, thioamide, or Q;

B is an alkyl, an alkenyl, an alkynyl, or an aryl linker, the alkyl oraryl portions of which is optionally substituted by an alkyl, analkenyl, an alkynyl, an aryl, an F, a CI, a Br, an I, a CN, an OH, analkoxy, an aryoxy, an alkylthio, an arylthio, a nitro, an azido, ahydrazino, a carboxyl, a thiocarboxyl, a carbonyl, a thiocarbonyl, acarboxylic acid ester, a thiocarboxylic acid ester, or an unsubstitutedor substituted amino, amide, thioamide, or Q; or

-   -   B in combination with A form an amide, a thioamide, a carboxylic        acid ester, a carboxylic acid thioester, a thiocarboxylic acid        ester, an imine, a hyrazone, or Q; and

Z is a reactive group comprising an isocyanate, an isothiocyanate, amonochlorotriazine, a dichlorotriazine, a 4,6-dichloro-1,3,5-triazines,a mono- or di-halogen substituted pyridine, a mono- or di-halogensubstituted diazine, a maleimide, a haloacetamide, an aziridine, asulfonyl halide, a carboxylic acid, an acid halide, a phosphonyl halide,a phosphoramidite (PO₂R¹⁴NR¹¹R¹²), a phosphoramidite thioanalogue(POSR¹⁴NR¹¹R¹²), a hydroxysuccinimide ester, a hydroxysulfosuccinimideester, an imido ester, an azido, a nitrophenol ester, an azide, a3-(2-pyridyl dithio)-propionamide, a glyoxal, an aldehyde, a thiol, anamine, a hydrazine, a hydroxyl, a terminal alkene, a terminal alkyne, aplatinum coordinate group or an alkylating agent.

In some of these embodiments, -A-B—Z is

where n is 1-10, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, for example 1-4.In certain of these embodiments, -A-B—Z is

providing a reactive hydroxysuccinimide ester group for coupling toamine moieties, as is known in the art. In more specific embodiments,-A-B—Z is

Some compounds of these embodiments comprise

In certain embodiments of those compounds, R⁶ and R⁹ are both H, or bothCH₃. In other embodiments of those compounds, X and Y are

-   -   (a) OH and O, respectively;    -   (b) NHCH₂CH₃ and NCH₂CH₃, respectively; or    -   (c) N(CH₃)₂ and N⁺(CH₃)₂, respectively.

Specific examples of the compounds of these embodiments comprise

Other specific examples comprise

Still other specific examples comprise

wherein E⁻ comprises an anion.

In some embodiments, any of the compounds described is a fluorescentdye.

Examples 1-16 below describe some of the methods available forsynthesizing several of the above dyes. Other methods are known in theart.

For purposes of synthesis of these dyes, reactive thiol, amine orhydroxyl groups can be protected during various synthetic steps and thereactive groups generated after removal of the protective group. Use ofa terminal alkene or alkyne groups for attachment of markers isdisclosed for example in U.S. Patent Publication 2003/0225247. The useof platinum coordinate groups for attachment of other dyes is disclosedfor example in U.S. Pat. No. 5,580,990, and the use of alkyl groups isdisclosed for example in U.S. Pat. No. 6,593,465.

In various embodiments, the dyes provided herein further comprise amember of a binding pair, to provide additional binding capabilities.The member of the binding pair can be covalently bound to any portion ofthe dye. In some of these embodiments, the member of a binding pair iscovalently bound to the fluorescent dye through reactive group Z.

Any binding pair now known or later discovered can be utilized in theseembodiments. Nonlimiting examples include sugar/lectins,antigen/antibodies, hapten/antibodies, ligand/receptors,hormone/receptors, enzyme/substrates, biotin/avidin, andbiotin/streptavidin.

Any one of the dyes of the instant invention can be utilized withanother dye to form a fluorescence energy transfer system, where thesignal is influenced by Förster resonance energy transfer (also known asfluorescence resonance energy transfer, or FRET). FRET uses twofluorophores (an energy transfer pair) where the emission spectrum ofone fluorophore (the donor) is of higher energy (having a shorterwavelength) and overlaps the absorption spectrum of the otherfluorophore (the acceptor). When the two fluorophores are brought withinabout 10-100 Å and the donor fluorophore is excited, the energy of thedonor is transferred to the acceptor by a resonance induceddipole-dipole interaction. This interaction is observed by fluorescencequenching of the donor fluorophore and/or emission of the acceptorfluorophore. FRET interactions are utilized with many assays,particularly in molecular biology. See, e.g., U.S. Pat. Nos. 4,868,103;5,237,515; and 6,117,635, U.S. Patent Publications 2005/0176014 and2005/0042618, and references cited therein.

Thus, in some embodiments, a fluorescence energy transfer system isprovided. The fluorescence energy transfer system comprises any of theabove-described fluorescent dyes and a second dye that is capable ofenergy transfer with the fluorescent dye. Such a system is utilized inExample 19, where PCR amplification of HCV RNA was performed with oneHCV primer labeled with Dye 1 (Example 4) as a FRET acceptor and anotherHCV primer labeled with fluorescein as a FRET donor. The primers areextended in the presence of HCV RNA and the extended primers hybridize,bringing the acceptor and donor dyes together to undergo a FRETinteraction.

In various embodiments, any of the fluorescent dyes described above isbound to a target molecule. In some of these embodiments, the dye iscovalently bound to the target molecule, e.g., by contacting reactivegroup Z with the target molecule such that reactive group Z reacts withthe target molecule to form a covalent bond between reactive group Z andthe target molecule. In other of these embodiments, the dye isnoncovalently bound to the target molecule, e.g., through a first memberof a binding pair on the target molecule and a second member of thebinding pair bound to the fluorescent dye through reactive group Z. Thislatter case is not narrowly limited to the use of any particular bindingpair. Nonlimiting examples of binding pair members that may be utilizedhere are sugars, lectins, antigens, haptens, antibodies, receptorsligands, hormone ligands, hormone receptors, enzymes, enzyme substrates,biotin, avidin, and streptavidin.

As used herein, a “target molecule” encompasses a moiety thatspecifically binds to an analyte. Thus, binding between theanalyte-specific moiety (“target”) and its corresponding analyte may bemonitored by essentially determining the presence or amount of dye thatis bound to the analyte. Examples of such assays include hybridizationsbetween complementary nucleic acids as well as binding betweenantibodies and their corresponding antigens. Other binding pairs thatmay be of interest include but are not limited to ligand/receptor,hormone/hormone receptor, antibody/antigen, carbohydrate/lectin andenzyme/substrate. Assays may be carried out where one component is fixedto a solid support and a corresponding partner is in solution. Bybinding to the component fixed to the support, the partner becomesattached to the support as well. A well-known example of this method ismicroarray assays where labeled analytes become bound to discrete siteson the microarray. Homogeneous probe-dependent assays are also wellknown in the art and may take advantage of the present invention.Examples of such methods are energy transfer between adjacent probes(U.S. Pat. No. 4,868,103), the Taqman exonuclease assay (U.S. Pat. Nos.5,538,848 and 5,210,015), Molecular Beacons (U.S. Pat. Nos. 5,118,801and 5,925,517) and various real time assays (U.S. Patent Publication2005/0137388).

These embodiments can utilize any target molecule now known or laterdiscovered. Examples of useful target molecules to which the dye can bebound include but are not limited to a nucleoside, nucleotide,oligonucleotide, polynucleotide, peptide nucleic acid, protein, peptide,enzyme, antigen, antibody, hormone, hormone receptor, cellular receptor,lymphokine, cytokine, hapten, lectin, avidin, streptavidin, digoxigenin,carbohydrate, oligosaccharide, polysaccharide, lipid, glycolipid, viralparticle, viral component, bacterial cell, bacterial component,eukaryotic cell, eukaryotic cell component, natural drug, syntheticdrug, glass particle, glass surface, natural polymers, syntheticpolymers, plastic particle, plastic surface, silicaceous particle,silicaceous surface, organic molecule, dyes and derivatives thereof.Where the target is a nucleoside, nucleotide, oligonucleotide, orpolynucleotide, such a target can comprise one or more ribonucleosidemoieties, ribonucleotide moieties, deoxyribonucleoside moieties,deoxyribonucleotide moieties, modified ribonucleosides, modifiedribonucleotides, modified deoxyribonucleosides, modifieddeoxyribonucleotides, ribonucleotide analogues, deoxyribonucleotideanalogues or any combination thereof.

The dyes of the present invention may have dyes as targets, therebycreating composite dyes. By joining the dyes of the present invention toanother dye, unique properties may be enjoyed that are not present ineither dye alone. For instance, if one of the dyes of the presentinvention is joined to another dye such that it creates an extendedconjugation system, the spectral characteristics of the dye may bedifferent than either dye component. Another example of this method iswhere the conjugation systems do not overlap but the proximity allows aninternal energy transfer to take place thereby extending the Stokesshift. See, e.g., U.S. Pat. Nos. 5,401,847; 6,008,373; and 5,800,996.Other properties may also be enhanced by this joining, for example, thejoining together of two ethidium bromide molecules generating a dye thathas enhanced binding to nucleic acids (U.S. Patent Publication2003/0225247). Other composite dyes have been described thatsimultaneously enjoy both properties, i.e. enhanced binding and energytransfer (U.S. Pat. No. 5,646,264). Furthermore, these composite dyesare not limited to binary constructs of only two dyes, but may compriseoligomeric or polymeric dyes. These composite dyes may be comprised ofthe same dye or different dyes may be joined together depending upon theproperties desired.

Antibodies labeled with dyes of the present invention may be used invarious formats. For example, an antibody with one of the dyes of thepresent invention may be used in an immunofluorescent plate assay or insitu analysis of the cellular location and quantity of various antigenicanalytes. Antibodies labeled with dyes may also be used free in solutionin cell counting or cell sorting methods that use a flow cytometer orfor in vitro or in vivo imaging of animal models. The presence orabsence of a signal may then be used to indicate the presence or absenceof the analyte itself. An example of this is a test where it issufficient to know whether a particular pathogen is present in aclinical specimen. Quantitative assays may also be carried out where theamount of target is being determined. An example of this is thepreviously cited microarray assay where the rise or fall in the amountof particular mRNA species may be of interest.

In another embodiment of the present invention, the dyes described abovemay be attached to a carrier with a more general affinity. Dyes may beattached to intercalators that in themselves do not provide signalgeneration but by virtue of their binding may bring a dye in proximityto a nucleic acid. A further example is attachment of dyes to SDSmolecules thereby allowing dyes to be brought into proximity toproteins. Thus this embodiment describes the adaptation of a dye or dyesthat lack affinity to a general class of molecules may be adapted bylinking them to non-dye molecules or macromolecules that can convey suchproperties. Various applications may enjoy the benefits of binding thedyes of the present invention to appropriate targets. As describedabove, staining of macromolecules in a gel is a methodology that has along history of use. More recent applications that also may find use arereal time detection of amplification (U.S. Pat. Nos. 5,994,056 and6,174,670, and U.S. Patent Publication 2005/0137388), and binding ofnucleic acids to microarrays. In situ assays may also find use where thebinding of dyes of the present invention is used to identify thelocation or quantity of appropriate targets.

The present invention also provides a kit for labeling a targetmolecule. The kit comprises any of the above-described fluorescent dyes,with additional reagents useful for labeling the target molecule. Thetarget molecule in these embodiments is not narrowly limited to anyparticular type of compound. Non-limiting examples include a nucleoside,nucleotide, oligonucleotide, polynucleotide, peptide nucleic acid,protein, peptide, enzyme, antigen, antibody, hormone, hormone receptor,cellular receptor, lymphokine, cytokine, hapten, lectin, avidin,streptavidin, digoxigenin, carbohydrate, oligosaccharide,polysaccharide, lipid, glycolipid, viral particle, viral component,bacterial cell, bacterial component, eukaryotic cell, eukaryotic cellcomponent, natural drug, synthetic drug, glass particle, glass surface,natural polymers, synthetic polymers, plastic particle, plastic surface,silicaceous particle, silicaceous surface, organic molecule, dyes andderivatives thereof. Where the target is a nucleoside, nucleotide,oligonucleotide, or polynucleotide, such a target can comprise one ormore ribonucleoside moieties, ribonucleotide moieties,deoxyribonucleoside moieties, deoxyribonucleotide moieties, modifiedribonucleosides, modified ribonucleotides, modifieddeoxyribonucleosides, modified deoxyribonucleotides, ribonucleotideanalogues, deoxyribonucleotide analogues or any combination thereof. Insome of these embodiments, the target molecule is a nucleic acid, anucleic acid analog, a protein, a peptide, an antibody, an antibodyfragment, a carbohydrate, a polysaccharide, an oligosaccharide, anucleotide, a nucleotide analog, a hapten, or an organic compound lessthan 2000 daltons. In particularly useful embodiments, the targetmolecule is a nucleic acid or a protein.

The additional reagents of these kits can include any reagents necessaryfor labeling any target molecule, such as a buffer, an enzyme, one orboth of a binding pair (as described above), chemical reagents to effectthe binding of the dye to the target molecule, and/or the targetmolecule itself. In some embodiments, the kit also includes instructionsfor labeling the target molecule.

Additionally provided is another kit for labeling a target molecule. Thekit in these embodiments comprises a first fluorescent dye and a secondfluorescent dye that form an energy transfer pair, wherein the firstfluorescent dye is any of the fluorescent dyes described above. In someembodiments, the kit also comprises additional reagents and/orinstructions useful for labeling target molecules with the energytransfer pair. The additional reagents of these kits can include anyreagents necessary for labeling any target molecule, such as a buffer,an enzyme, one or both of a binding pair (as described above), chemicalreagents to effect the binding of the dye to the target molecule, and/orthe target molecule itself.

As with the previously described kits, the target molecule in theseembodiments is not narrowly limited to any particular type of compound,and could include, e.g., any of the target molecules discussedpreviously. In some embodiments, the target molecule is a nucleic acidor a protein.

The present invention is also directed to a target molecule labeled withany of the fluorescent dyes described above.

The target molecule in these embodiments is not narrowly limited to anyparticular type of compound. Non-limiting examples include a nucleoside,nucleotide, oligonucleotide, polynucleotide, peptide nucleic acid,protein, peptide, enzyme, antigen, antibody, hormone, hormone receptor,cellular receptor, lymphokine, cytokine, hapten, lectin, avidin,streptavidin, digoxigenin, carbohydrate, oligosaccharide,polysaccharide, lipid, glycolipid, viral particle, viral component,bacterial cell, bacterial component, eukaryotic cell, eukaryotic cellcomponent, natural drug, synthetic drug, glass particle, glass surface,natural polymers, synthetic polymers, plastic particle, plastic surface,silicaceous particle, silicaceous surface, organic molecule, dyes andderivatives thereof. Where the target is a nucleoside, nucleotide,oligonucleotide, or polynucleotide, such a target can comprise one ormore ribonucleoside moieties, ribonucleotide moieties,deoxyribonucleoside moieties, deoxyribonucleotide moieties, modifiedribonucleosides, modified ribonucleotides, modifieddeoxyribonucleosides, modified deoxyribonucleotides, ribonucleotideanalogues, deoxyribonucleotide analogues or any combination thereof. Insome of these embodiments, the target molecule is a nucleic acid, anucleic acid analog, a protein, a peptide, an antibody, an antibodyfragment, a carbohydrate, a polysaccharide, an oligosaccharide, anucleotide, a nucleotide analog, a hapten, or an organic compound lessthan 2000 daltons. In particularly useful embodiments, the targetmolecule is a nucleic acid or a protein.

In some of these embodiments, the fluorescent dye is covalently bound tothe target molecule, for example through reactive group Z.

In other embodiments, the fluorescent dye is noncovalently bound to thetarget molecule, for example through a binding pair, e.g., where onemember of the binding pair is covalently bound to the dye throughreactive group Z and the other member of the binding pair is covalentlybound to the target, by any means known in the art. The binding pair inthese embodiments can be any binding pair now known or later discovered.Nonlimiting examples include a sugar/lectin, an antigen/antibody, ahapten/antibody, a ligand/receptor, a hormone/receptor, anenzyme/substrate, biotin/avidin, or biotin/streptavidin.

In some of these embodiments, the labeled target molecule furthercomprises a second dye such that the second dye forms an energy transferpair with the fluorescent dye. Examples of such compositions are wellknown in the art. See, e.g., U.S. Patent Publication 2005/0137388,describing nucleic acids labeled with both a donor and an acceptor dye.

The labeled target molecule of these embodiments can also be part of acomposition that further comprises a second labeled target molecule,where the label on the labeled target molecule and the label on thesecond labeled target molecule form an energy transfer pair. Examplesinclude two labeled primers, where the two labels form an energytransfer pair, or an antibody labeled with one member of an energytransfer pair and the corresponding antigen labeled with the othermember of the energy transfer pair. See, e.g., U.S. Patent Publication2005/0137388, PCT Publication WO99/47700 and U.S. Pat. Nos. 5,237,515and 4,868,103.

In further embodiments, the invention is directed to a method oflabeling a target molecule. The method comprises contacting reactivegroup Z of any of the above-described fluorescent dyes with the targetmolecule such that reactive group Z reacts with the target molecule toform a covalent bond between reactive group Z and the target molecule.

The target molecule in these embodiments is not narrowly limited to anyparticular type of compound. Non-limiting examples include a nucleoside,nucleotide, oligonucleotide, polynucleotide, peptide nucleic acid,protein, peptide, enzyme, antigen, antibody, hormone, hormone receptor,cellular receptor, lymphokine, cytokine, hapten, lectin, avidin,streptavidin, digoxigenin, carbohydrate, oligosaccharide,polysaccharide, lipid, glycolipid, viral particle, viral component,bacterial cell, bacterial component, eukaryotic cell, eukaryotic cellcomponent, natural drug, synthetic drug, glass particle, glass surface,natural polymers, synthetic polymers, plastic particle, plastic surface,silicaceous particle, silicaceous surface, organic molecule, dyes andderivatives thereof. Where the target is a nucleoside, nucleotide,oligonucleotide, or polynucleotide, such a target can comprise one ormore ribonucleoside moieties, ribonucleotide moieties,deoxyribonucleoside moieties, deoxyribonucleotide moieties, modifiedribonucleosides, modified ribonucleotides, modifieddeoxyribonucleosides, modified deoxyribonucleotides, ribonucleotideanalogues, deoxyribonucleotide analogues or any combination thereof. Insome of these embodiments, the target molecule is a nucleic acid, anucleic acid analog, a protein, a peptide, an antibody, an antibodyfragment, a carbohydrate, a polysaccharide, an oligosaccharide, anucleotide, a nucleotide analog, a hapten, or an organic compound lessthan 2000 daltons. In particularly useful embodiments, the targetmolecule is a nucleic acid or a protein.

In some of these embodiments, the target molecule further comprises asecond dye such that the fluorescent dye and the second dye form anenergy transfer pair.

The present invention further provides another method of labeling atarget molecule. In these embodiments, the method comprises contactingany of the above-described fluorescent dyes with the target molecule,wherein the target molecule comprises a second member of the bindingpair. The dye in these embodiments comprises the first member of thebinding pair. As such, when the dye is combined with the targetmolecule, the first and second members of the binding pair bindtogether, thus noncovalently labeling the target molecule with the dye.

These embodiments encompass the use of any target molecule now known orlater discovered, e.g., as described above. In some embodiments, thetarget molecule is a nucleic acid, a nucleic acid analog, a protein, apeptide, an antibody, an antibody fragment, a carbohydrate, apolysaccharide, an oligosaccharide, a nucleotide, a nucleotide analog, ahapten, or an organic compound less than 2000 daltons. In particularlyuseful embodiments, the target molecule is a nucleic acid or a protein,as described above.

As with above-described embodiments, any binding pair now known or laterdiscovered can be utilized for these methods. Nonlimiting examples ofuseful binding pairs are a sugar/lectin, an antigen/antibody, ahapten/antibody, a ligand/receptor, a hormone/receptor, anenzyme/substrate, biotin/avidin, or biotin/streptavidin.

Preferred embodiments are described in the following examples. Otherembodiments within the scope of the claims herein will be apparent toone skilled in the art from consideration of the specification orpractice of the invention as disclosed herein. It is intended that thespecification, together with the examples, be considered exemplary only,with the scope and spirit of the invention being indicated by theclaims, which follow the examples.

Example 1. Synthesis of 7-methoxy-2,2,4-trimethyl-1,2-dihydroquinoline

The compound m-anisidine (26 ml, 0.23 mol) was slowly added to aceticacid (2.6 ml) with stirring, followed by slow addition of mesityl oxide(27 ml, 0.23 mol). After the mixture was stirred at room temperatureovernight, concentrated hydrobromic acid (50 ml) was added. The mixturewas stirred for an additional hour. The precipitate was then filteredand washed with acetone. The resulting solid was then dissolved in water(100 ml) and neutralized to pH 7 with 10N aqueous sodium hydroxide. Theresulting solution was extracted with chloroform (3×50 mL) and driedover anhydrous sodium sulfate. After filtering off the sodium sulfate,the solution was evaporated under vacuum to give crude product, whichwas recrystalized with hexanes to give a yellowish solid (15.5 g, 33%yield). The structure of 7-methoxy-2,2,4-trimethyl-1,2-dihydroquinolineis:

Example 2. Synthesis of1-ethyl-7-methoxy-2,2,4-trimethyl-1,2-dihydroquinoline

The compound 7-methoxy-2,2,4-trimethyl-1,2-dihydroquinoline (5.0 g, 24.6mmols) from Example 1 was dissolved in anhydrous DMF (40 ml). Calciumcarbonate (3.0 g, 30 mmols) and ethyl iodide (4.7 g, 30 mmols) weresubsequently added. The mixture was heated at 120° C. with vigorousstirring for 18 hours. After the mixture was cooled to room temperature,it was poured into water (300 mL). The suspension was filtered through apad of celite then extracted with chloroform (3×100 mL). The combinedchloroform layer was washed with water (3×200 mL) and then dried withanhydrous sodium sulfate. The solvent was evaporated under vacuum togive a dark green oil (5.72 g, 100% yield). The structure of1-ethyl-7-methoxy-2,2,4-trimethyl-1,2-dihydroquinoline is:

Example 3. Synthesis of 1-ethyl-2,2,4-trimethyl-12-dihydroquinolin-7-ol

The compound 1l-ethyl-7-methoxy-2,2,4-trimethyl-1,2-dihydroquinoline(5.72 g) from Example 2 was added to a mixture of concentratedhydrobromic acid (13 mL) and glacial acetic acid (13 mL). After themixture was stirred at reflux for 6 hours, it was cooled with ice andneutralized with 10 N aqueous sodium hydroxide to pH 7. The mixture wasthen extracted with chloroform (3×50 ml) and dried over anhydrous sodiumsulfate, then filtered and evaporated to give a sticky green oil as thecrude product (6.02 g), which was used without further purification. Thestructure of 1-ethyl-2,2,4-trimethyl-1,2-dihydroquinolin-7-ol is givenbelow:

Example 4. Synthesis of Dye 1 (EZRed620)

The compounds 1-ethyl-2,2,4-trimethyl-1,2-dihydroquinolin-7-ol (220 mg,1.0 mmol) (Example 3) and 2-(4-formylphenoxy)acetic acid (61 mg, 0.34mmol) were mixed thoroughly and heated at 150° C. with vigorous stirringfor 15 min in a microwave reactor. After the mixture was cooled to roomtemperature, methanol (5%) in chloroform (total 5 ml) was added,followed by the addition of tetrachloro-1,4-benzoquinone (25.5 mg, 0.51mmol). This mixture was stirred at room temperature for 20 min. Thesolvent was then removed under vacuum and the residue purified by flashchromatography. The solvent was removed to give Dye 1 (shown below) as adark solid (32.2 mg, yield: 16%). λ_(abs)=594 nm (in methanol),λ_(em)=611 nm (in methanol).

Example 5. Synthesis of Dye 2

Dye 2 (shown below) was prepared using the procedure described inExample 4 except that 1-ethyl-1,2,3,4-tetrahydroquinolin-7-olsubstituted for 1-ethyl-2,2,4-trimethyl-1,2-dihydroquinolin-7-ol. Yield:25%. λ_(abs)=558 nm (in methanol), λ_(em)=574 nm (in methanol).

Example 6. Synthesis of2-(4-(3-hydroxy-6-oxo-6H-xanthen-9-yl)phenoxy)acetic acid (Dye 3)

Dye 3 (shown below) was prepared using the procedure described inExample 4 except that 2-(4-formylphenoxy)acetic acid and resorcinolsubstituted for 1-ethyl-2,2,4-trimethyl-1,2-dihydroquinolin-7-ol and2-(4-formylphenoxy)acetic acid. Yield: 34%. λ_(abs)=485 nm (inmethanol), λ_(em)=511 nm (in methanol).

Example 7. Synthesis of2-(4-(3-(ethylamino)-6-(ethylimino)-2,7-dimethyl-6H-xanthen-9-yl)phenoxy)aceticacid (Dye 4)

Dye 4 (shown below) was prepared using the procedure described inExample 4 except that 3-(ethylamino)-4-methylphenol and2-(4-formylphenoxy)acetic acid substituted for1-ethyl-2,2,4-trimethyl-1,2-dihydroquinolin-7-ol and2-(4-formylphenoxy)acetic acid. Yield: 12%. λ_(abs)=525 nm (inmethanol), λ_(em)=540 nm (in methanol).

Example 8. Synthesis of Dye 5

Dye 5 (shown below) was prepared using the procedure described inExample 4 except that 2-(4-formylphenoxy)acetic acid and8-hydroxyjulolidine substituted for1-ethyl-2,2,4-trimethyl-1,2-dihydroquinolin-7-ol and2-(4-formylphenoxy)acetic acid. Yield: 22%. λ_(abs)=570 nm (inmethanol), λ_(em)=584 nm (in methanol).

Example 9. Synthesis ofN-(9-(4-(carboxymethoxy)phenyl)-6-(dimethylamino)-3H-xanthen-3-ylidene)-N-methylmethanaminium(Dye 6)

Dye 6 (shown below) was prepared using the procedure described inExample 4 except that 2-(4-formylphenoxy)acetic acid and3-(dimethylamino)phenol substituted for1-ethyl-2,2,4-trimethyl-1,2-dihydroquinolin-7-ol and2-(4-formylphenoxy)acetic acid. Yield: 36%. λ_(abs)=548 nm (inmethanol), λ_(em)=566 nm (in methanol).

Example 10. Synthesis of 5-(4-formylphenoxy)pentanoic acid

Methyl 5-bromovelerate (13.4 g, 68.6 mmol) and anhydrous potassiumcarbonate (18.93 g, 137 mmol) was added to a solution of4-hydroxybenzaldehyde (8.38 g, 68.6 mmol) in anhydrous acetone (140 ml).The mixture was heated at reflux for 16 hours with vigorous stirring.After the mixture was cooled to room temperature and filtered, thesolvent was removed under vacuum. The residue was dissolved indichloromethane (200 mL) and washed sequentially with aqueous sodiumhydroxide (1 N, 200 ml), water (200 ml) and brine (200 ml). The solventwas evaporated under vacuum to give a yellowish crystal. The crystal wasdissolved in a mixture of THF (200 ml) and hydrochloric acid (6 N, 30ml). The mixture was then heated to reflux for 3 hours, after which theTHF was removed under vacuum. The oil was then extracted with chloroform(4×50 ml). The combined chloroform layer was washed with water (2×150ml) and brine (200 ml), and then dried with anhydrous sodium sulfate.After the solvent was removed, the acid was obtained as a yellow liquid.The structure of 5-(4-formylphenoxy)pentanoic acid is:

Example 11. Synthesis of Dye 7

Dye 7 (shown below) was prepared using the procedure described inExample 4 except that 5-(4-formylphenoxy)pentanoic acid and1-ethyl-2,2,4-trimethyl-1,2-dihydroquinolin-7-ol substituted for1-ethyl-2,2,4-trimethyl-1,2-dihydroquinolin-7-ol and2-(4-formylphenoxy)acetic acid. Yield: 17%. λ_(abs)=590 nm (inmethanol), λ_(em)=613 nm (in methanol).

Example 12. Synthesis of Dye 8

Dye 8 (shown below) was prepared using the procedure described inExample 4 except that 5-(4-formylphenoxy)pentanoic acid and1-ethyl-1,2,3,4-tetrahydroquinolin-7-ol substituted for1-ethyl-2,2,4-trimethyl-1,2-dihydroquinolin-7-ol and2-(4-formylphenoxy)acetic acid. Yield: 23%. λ_(abs)=559 nm (inmethanol), λ_(em)=574 nm (in methanol).

Example 13. Synthesis of5-(4-(3-hydroxy-6-oxo-6H-xanthen-9-yl)phenoxy)pentanoic acid (Dye 9)

Dye 9 (shown below) was prepared using the procedure described inExample 4 except that 5-(4-formylphenoxy)pentanoic acid and resorcinolsubstituted for 1-ethyl-2,2,4-trimethyl-1,2-dihydroquinolin-7-ol and2-(4-formylphenoxy)acetic acid. Yield: 26%. λ_(abs)=486 nm (inmethanol), λ_(em)=513 nm (in methanol).

Example 14. Synthesis of(Z)-5-(4-(3-(ethylamino)-6-(ethylimino)-2,7-dimethyl-6H-xanthen-9-yl)phenoxy)pentanoicacid (Dye 10)

Dye 10 (shown below) was prepared using the procedure described inExample 4 except that 3-(ethylamino)-4-methylphenol and5-(4-formylphenoxy)pentanoic acid substituted for1-ethyl-2,2,4-trimethyl-1,2-dihydroquinolin-7-ol and2-(4-formylphenoxy)acetic acid. Yield: 17%. λ_(abs)=524 nm (inmethanol), λ_(em)=541 nm (in methanol).

Example 15. Synthesis of Dye 11

Dye 11 (shown below) was prepared using the procedure described inExample 4 except that 5-(4-formylphenoxy)pentanoic acid and8-hydroxyjulolidine substituted for1-ethyl-2,2,4-trimethyl-1,2-dihydroquinolin-7-ol and2-(4-formylphenoxy)acetic acid. Yield: 28%. λ_(abs)=569 nm (inmethanol), λ_(em)=584 nm (in methanol). The structure of dye 11 is givenbelow:

Example 16. Synthesis ofN-(9-(4-(4-carboxybutoxy)phenyl)-6-(dimethylamino)-3H-xanthen-3-ylidene)-N-methylmethanaminium(Dye 12)

Dye 12 (shown below) was prepared using the procedure described inExample 4 except that 5-(4-formylphenoxy)pentanoic acid and3-(dimethylamino)phenol substituted for1-ethyl-2,2,4-trimethyl-1,2-dihydroquinolin-7-ol and2-(4-formylphenoxy)acetic acid. Yield: 23%. λ_(abs)=547 nm (inmethanol), λ_(em)=565 nm (in methanol). The structure of dye 12 is givenbelow:

Example 17. Comparison of Spectra of Dye 1 (EZRed620) with Roche LC-Red640 Dye

Spectral properties of Dye 1 (Example 4) were determined and compared toRoche LC-Red 640. The UV-Vis spectra of EZRed620 and LC Red 640 wererecorded in methanol with a NanoDrop® ND-1000 Spectrophotometer. Thewavelengths of maximum absorption of EZRed620 and LC Red 640 were at 589nm and 613 nm, respectively. The fluorescence spectra were recorded inmethanol with Photo Technology International (PTI) fluorometer. Themaximum emission wavelengths of EZRed620 and LC Red 640 were 611 nm and637 nm under these conditions, respectively. Graphs of the results ofthese studies are shown in FIGS. 1A-1D.

Example 18. Bioconjugation of Dye 1 to Oligonucleotide

Dye 1 (2 μmol) was dissolved in amine-free DMF (140 μl), followed by theaddition of 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate(2.4 μmols) and diisopropylethylamine (4.4 μmols). The mixture wasstirred at room temperature for 30 min, and then added to a solution ofoligonucleotide containing an amine linker (80 nmols) in 0.9 M sodiumborate buffer (320 μL, pH 8.5). The mixture was stirred at roomtemperature for 16 h. The solvent was removed under vacuum and theresidue pellet was purified by HPLC using a gradient oftriethylammoniumacetate (0.1 M, pH 6.9) and acetonitrile as elutingsolvents. The fractions containing pure conjugates were combined andevaporated, and then coevaporated with water to remove excessive salt.The final blue pellet was dissolve in water and stored at −20° C.

Example 19. HCV Test with Dye 1 (EZRed620)

RNA was isolated from 400 μl plasma or serum from each sample using aQIAamp MinElute Virus Spin Kit in a QIAcube system (QIAGEN, ValenciaCalif.) according to the manufacturer's protocol. The resulting RNA waseluted in 50 μl elution buffer. Five μl of the eluate was subjected toRT-PCR to amplify the HCV target sequenceGAGGAACUACUGUCUUCACGCAGAAAGCGUCUAGCCAUGGCGUUAGUAUGAGUG UCG (SEQ IDNO:1). The PCR forward primer was GAGGAACTACTGTCTTCACGCAGAAAGCG (SEQ IDNO:2); the reverse primer was CGACACTCATACTAACGCCATGGCTAG (SEQ ID NO:3).The forward primer was labeled on the underlined/bolded C with Dye 1(EZRed620) as a FRET acceptor; the reverse primer was labeled on theunderlined/bolded T with fluorescein as a FRET donor. Reversetranscription and PCR amplification was carried out in either a RocheLight Cycler or a Qiagen Rotor-Gene Q RealTime PCR machine. Reversetranscription was performed at 50° C. for 30 min. PCR amplification wasconducted at 95° C. for 15 sec to denature and 66° C. for 60 sec. forannealing/extension, with a total of 50 or 60 cycles. RealTime RT-PCRprogress was monitored through measuring the strength of the EZRed620signal. When the Roche Light Cycler system was used, Channel F2 waschosen to measure the EZRed620 signal; when the Qiagen Rotor-gene Qsystem was used, 470 nm was used for excitation and either 610 nm or 660nm was used to measure the EZRed620 emission.

Comparison of EZRed620/Rotor-Gene Q with LCRed640/COBAS AmpliPrep forDetermination of HCV Viral Load in Clinical Samples

Sixty samples, numbered 101 to 160, were tested as positive using theRoche COBAS AmpliPrep. Those samples were subsequently tested using (a)the EZRed620/Rotor-Gene Q system as described above and (b) theLCRed640/COBAS system as per the manufacturer's instructions. Fouradditional samples, numbered 201 to 204, were also tested. Sample 204was tested negative with both platforms. All samples were run withnegative control samples of water or elution buffer and a known positivesample. Results of the HCV viral load determination for theabove-described samples using both platforms is provided in Table 1. Thecovariance between the EZRed620/Rotor-Gene Q system and theLCRed640/COBAS system was 1.077, r=0.95. This shows that theEZRed620/Rotor-Gene Q system can reliably substitute for theLCRed640/COBAS system with similar results.

TABLE 1 Comparison of RT-PCR HIV viral load determination using twosystems. EZRed620 LCRed640 Rotor-Gene COBAS Sample # Log₁₀ Log₁₀ 1016.55 6.62 102 6.36 6.02 103 5.14 5.06 104 6.22 6.43 105 6.02 5.29 1066.37 6.85 107 6.21 5.94 108 6.49 6.25 109 6.29 6.10 110 6.35 5.68 1214.41 3.84 122 6.35 6.71 123 2.67 2.02 124 4.85 4.29 125 7.09 7.58 1264.40 4.94 127 6.26 6.36 128 6.02 5.80 129 6.30 6.92 130 5.08 4.81 1315.76 5.48 132 5.36 4.99 133 6.08 5.85 134 6.83 7.26 135 6.73 6.88 1366.78 7.61 137 6.02 6.03 138 6.54 6.96 139 6.54 6.86 140 5.34 5.23 1414.37 4.83 142 2.86 3.22 143 5.59 5.12 144 6.23 5.91 145 5.59 4.95 1466.34 6.22 147 5.62 5.64 148 7.02 6.95 149 6.76 6.56 150 6.95 6.85 1516.60 7.01 152 5.53 5.25 153 6.60 6.60 154 6.46 6.24 155 6.61 6.42 1566.05 6.06 157 6.81 6.73 158 5.62 5.40 159 5.03 4.96 160 6.84 7.45 2016.31 6.26 202 5.56 5.40 203 3.12 3.02

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In view of the above, it will be seen that several objectives of theinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references.

What is claimed is:
 1. A method for fluorescently labeling a targetmolecule, comprising the steps of: providing a fluorescent dye compoundhaving the formula:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are independently H,F, Cl, Br, I, CN, nitro, azido, hydroxyl, amino, hydrazino,(substituted) aryl, (substituted) aryloxy, alkenyl, alkynyl, alkyl,alkoxy, alkylamino, dialkylamino, arylamino, diarylamino,alkyl(aryl)amino, alkanoylamino, alkylthio, alkylcarbonyl, arylcarbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkyloxycarbonyl,aroxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl, alkyl(aryl)aminocarbonyl,arylcarboxamido, or Q, the alkyl or alkoxy portions of which aresaturated or unsaturated, linear or branched, unsubstituted or furthersubstituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl,amide, thioamide, or Q; wherein Q is selected from the group consistingof a carboxyl group (CO₂ ⁻), a carbonate ester (COER¹¹), a sulfonateester (SO₂ER¹¹), a sulfoxide (SOR¹¹), a sulfone (SO₂CR¹¹R¹²R¹³), asulfonamide (SO₂NR¹¹R¹²), a phosphate (PO₄ ⁻), a phosphate monoester(PO₃ ⁻ER¹¹), a phosphate diester (PO₂ER¹¹ER¹²), a phosphonate (PO₃ ⁻), aphosphonate monoester (PO₂ ⁻ER¹¹), a phosphonate diester (POER¹¹ER¹²), athiophosphate (PSO₃ ⁻), a thiophosphate monoester (PSO₂ ⁻ER¹¹), athiophosphate diester (PSOER¹¹ER¹²), a thiophosphonate (PSO₂ ⁻), athiophosphonate monoester (PSO⁻ER¹¹), a thiophosphonate diester(PSER¹¹ER¹²), a phosphonamide (PONR¹¹R¹²NR¹⁴R¹⁵), a phosphonamidethioanalogue (PSNR¹¹R¹²NR¹⁴R¹⁵), a phosphoramide (PONR¹¹R¹²NR¹³NR¹⁴R¹⁵),a phosphoramide thioanalogue (PSNR¹¹R¹²NR¹³NR¹⁴R¹⁵S), a phosphoramidite(PO₂R¹⁴NR¹¹R¹²), and a phosphoramidite thioanalogue (POSR¹⁴NR¹¹R¹²),wherein E can independently be O or S, and wherein the aryl portions ofany of the above are optionally substituted by F, Cl, Br, I, CN, OH,alkenyl, alkynyl, alkylcarbonyl, amide, or thioamide; wherein R¹ incombination with R², R³ in combination with R⁴, R⁵ in combination withR⁶, or R⁹ in combination with R¹⁰ can independently form a 5-10 memberring structure which is saturated or unsaturated, and which isoptionally further substituted with an alkyl, an aryl, an alkenyl, analkynyl, an alkoxy, an aryloxy, a hydroxyl, F, Cl, Br, I, CN, a nitro,an alkylsulfonyl, an arylsulfonyl, an alkylsulfinyl, an arylsulfinyl, a(thio)carbonyl, a (thio)carboxylic acid, a (thio)carboxylic acid ester,a nitro, an amino, a (thio)amide, an azido, a hydrazino, or a(thio)phosphonate where each alkyl group or alkoxy group isindependently saturated or unsaturated, linear or branched, orsubstituted or unsubstituted and each aryl group wherein isindependently optionally substituted with F, Cl, Br, I, CN, OH, analkyl, an alkenyl, an alkynyl, an alkoxy, an aryoxy, an alkylthio, anarylthio, a nitro, an azido, a hydrazino, a carboxyl, a thiocarboxyl, acarbonyl, a thiocarbonyl, a carboxylic acid ester, a thiocarboxylic acidester, or an amino, amide, thioamide, or Q; R¹¹, R¹², R¹³, R¹⁴ and R¹⁵are independently hydrogen, a halogen, an amino group, an alkyl groupwherein said alkyl group is saturated or unsaturated, linear orbranched, or substituted or unsubstituted, an alkoxy group wherein saidalkoxy group is saturated or unsaturated, branched or linear, orsubstituted or unsubstituted, an aryl group wherein said aryl group isunsubstituted or substituted; wherein R¹¹ in combination with R¹², R¹⁴in combination with R¹⁵, R¹¹ in combination with R¹³, R¹¹ in combinationwith R¹⁴, R¹² in combination with R¹⁵, or R¹³ in combination with R¹⁴can independently form a 5-10 member ring; X is O, OR¹⁶, NR¹⁷R¹⁸ orN⁺R¹⁷R¹⁸; Y is O, OR¹⁶, NR¹⁹R²⁰ or N⁺R¹⁹R²⁰, wherein R¹⁶, R¹⁷, R¹⁸, R¹⁹and R²⁰ are independently H, alkyl, alkenyl, alkynyl, or aryl; or R¹⁷ incombination with R¹⁸, or R¹⁹ in combination with R²⁰ can independentlyform a 5-10 member ring structure which is optionally furthersubstituted with alkyl, alkenyl, alkynyl, aryl, alkoxy, F, Cl, Br, I,carboxylic acid or carboxylic acid ester, where the alkyl group issaturated or unsaturated, linear or branched, and is optionally furthersubstituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, nitro, azido,hydrazino, alkoxy, aryoxy, alkylthio, arylthio, thiocarboxyl, carbonyl,thiocarbonyl, thiocarboxylic acid ester, amino, amide, thioamide, or Q,and the aryl group wherein is optionally substituted by F, Cl, Br, I,CN, OH, alkoxy, aryoxy, alkylthio, arylthio, nitro, azido, hydrazino,carboxyl, thiocarboxyl, carbonyl, thiocarbonyl, carboxylic acid ester,thiocarboxylic acid ester, amino, amide, thioamide, or Q; wherein R¹⁷ incombination with R⁶, R¹⁸ in combination with R⁷, R¹⁹ in combination withR⁸, and R²⁰ in combination with R⁹, can independently form a 5- to10-member ring structure that is saturated or unsaturated and optionallyfurther substituted with an alkyl, an aryl, an alkenyl, an alkynyl, analkoxy, an aryloxy, a hydroxyl, F, Cl, Br, I, CN, a nitro, a carbonyl, athiocarbonyl, a thiocarboxylic acid, a thiocarboxylic acid ester, anitro, an amino, a (thio)amide, an azido, a hydrazino, or Q, wherein thealkyl group herein is saturated or unsaturated, linear or branched,substituted or unsubstituted, an alkoxy group wherein the alkoxy groupis saturated or unsaturated, branched or linear, substituted orunsubstituted; and wherein the aryl group is optionally substituted withF, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkoxy, aryoxy, alkylthio,arylthio, nitro, azido, hydrazino, carboxyl, thiocarboxyl, carbonyl,thiocarbonyl, carboxylic acid ester, thiocarboxylic acid ester, amino,amide, thioamide, or Q; A is O, S or NR²¹, wherein R²¹ is a hydrogen, analkyl, an aryl, an alkenyl, an alkynyl, an alkylcarbonyl, anarylcarbonyl, an alkylaminocarbonyl, or an arylaminocarbonyl, the alkylor aryl portions of which is optionally substituted by an alkyl, anaryl, an alkenyl, an alkynyl, F, Cl, Br, I, CN, OH, an alkoxy, anaryoxy, an alkylthio, an arylthio, a nitro, an azido, a hydrazino, athiocarboxyl, a carbonyl, a thiocarbonyl, a thiocarboxylic acid ester,or an amino, amide, thioamide, or Q; B is an alkyl, an alkenyl, analkynyl, or an aryl linker, the alkyl or aryl portions of which isoptionally substituted by an alkyl, an alkenyl, an alkynyl, an aryl, F,Cl, Br, I, CN, OH, an alkoxy, an aryoxy, an alkylthio, an arylthio, anitro, an azido, a hydrazino, a carboxyl, a thiocarboxyl, a carbonyl, athiocarbonyl, a carboxylic acid ester, a thiocarboxylic acid ester, oran amino, amide, thioamide, or Q; or B in combination with A form anamide, a thioamide, a carboxylic acid ester, a carboxylic acidthioester, a thiocarboxylic acid ester, an imine, a hyrazone, or Q; andZ is a reactive group selected from the group consisting of anisocyanate, an isothiocyanate, a monochlorotriazine, a dichlorotriazine,a 4,6-dichloro-1,3,5-triazines, a mono- or di-halogen substitutedpyridine, a mono- or di-halogen substituted diazine, a maleimide, ahaloacetamide, an aziridine, a sulfonyl halide, a carboxylic acid, anacid halide, a phosphonyl halide, a phosphoramidite (PO₂R¹⁴NR¹¹R¹²), aphosphoramidite thioanalogue (POSR¹⁴NR¹¹R¹²), a hydroxysuccinimideester, a hydroxysulfosuccinimide ester, an imido ester, an azido, anitrophenol ester, an azide, a 3-(2-pyridyl dithio)-propionamide, aglyoxal, an aldehyde, a thiol, an amine, a hydrazine, a hydroxyl, aterminal alkene, a terminal alkyne, a platinum coordinate group and analkylating agent, wherein the carbon lengths for said alkenyl, alkynyl,and alkyl groups are in the range of 1 to 16; providing a targetmolecule to be labeled, wherein the target molecule is selected from thegroup consisting of a nucleic acid, a nucleotide, and a peptide nucleicacid; and contacting the fluorescent dye compound with the targetmolecule such that reactive group Z reacts with the target molecule toform a covalent bond between reactive group Z and the target molecule.2. The method of claim 1, wherein -A-B—Z is

wherein n is 1-10.
 3. The method of claim 2, wherein n is 1-4.
 4. Themethod of claim 2, wherein -A-B—Z is


5. The method of claim 1, wherein -A-B—Z is


6. The method of claim 1, wherein the fluorescent dye compound has thestructure


7. The method of claim 6, wherein R⁶ and R⁹ are both H or CH₃.
 8. Themethod of claim 6, wherein X and Y are (a) OH and O, respectively; (b)NHCH₂CH₃ and NCH₂CH₃, respectively; or (c) N(CH₃)₂ and N⁺(CH₃)₂,respectively.
 9. The method of claim 1, wherein the fluorescent dyecompound is selected from the group consisting of:


10. The method of claim 9, wherein the fluorescent dye compound isselected from the group consisting of:


11. The method of claim 1, wherein the fluorescent dye compound isselected from the group consisting of:


12. The method of claim 1, wherein the fluorescent dye compound isselected from the group consisting of:

wherein E⁻ is an anion.
 13. The method of claim 1, wherein the targetmolecule is a nucleic acid.
 14. The method of claim 1, wherein thetarget molecule is a peptide nucleic acid.
 15. The method of claim 1,wherein the target molecule is a nucleotide.
 16. The method of claim 1,wherein the carbon lengths for said alkenyl, alkynyl, and alkyl groupsare in the range of 1 to
 10. 17. The method of claim 13, wherein thecarbon lengths for said alkenyl, alkynyl, and alkyl groups are in therange of 1 to 10.